Electrophoretic separations of such species as DNA, RNA or proteins in slab gels are generally followed by the blotting or immobilization of the separated species onto the surface of a treated paper or membrane The zone pattern of the separated species is maintained during the blotting procedure, and the blotting permits one to preserve the zone pattern or to separate and isolate the individual zones, thus serving an important function for both analytical and preparative purposes
Separation and blotting are commonly performed in two separate pieces of apparatus, requiring a certain amount of manual manipulation by the user. Capillary blotting, based on the studies reported by Southern, E.M., J. Mol. Biol., 98. 503 (1975), relies on the liquid absorbent character of filter papers and similar materials to draw buffer solution from one side of the gel to the other, the buffer solution drawing the solute zones along with it. The solute zones are immobilized by the treated paper or membrane upon contact, resisting further movement Vacuum transfer is an alternative to capillary blotting, and uses a pressure differential to cause the bulk migration of buffer solution across the gel. The mechanism by which the solute zones are extracted from the gel and immobilized on the paper or membrane is the same as in capillary blotting. The third known alternative is electrophoretic blotting, where the transfer is performed electrophoretically by imposing an electric potential in the direction perpendicular to the plane of the gel.
Each of these techniques has inherent advantages. In capillary blotting, the speed and quality of the transfer depend upon the user's ability to avoid air bubbles in the liquid migration path. In addition, capillary blotting is a slow procedure, generally requiring 8-24 hours unless the absorbing sheets at the downstream side of the flow direction are periodically replaced. Even then, capillary transfers generally require a minimum of about 4 hours. A vacuum transfer generally requires 2-4 hours, plus an additional hour to perform a series of gel soaks for various purposes prior to the application of the vacuum. Electrophoretic transfer is the fastest of the three, permitting transfers in as little as ten minutes. Each of these procedures, however, involves removing the gel from the separation cell and physically placing it in the blotting apparatus, which steps are performed manually by the user. Gels are very delicate, and handling invariably raises a risk of damage to the gel. In addition, the physical transfer of the gel between the two pieces of apparatus is time consuming.
An apparatus has now been developed which permits electrophoretic separation in a slab gel followed by transfer of the separated zone pattern to an immobilizing sheet or membrane with no intervening movement of the slab gel required. The electrophoretic separation is performed in a submarine-type cell, equipped with additional electrodes appropriately placed for electrophoretic transfer of the separated zones in the direction normal to the gel once the separation is complete. The gel is arranged horizontally in the cell where it is submerged in buffer solution between a pair of elongate separation electrodes arranged to cause sample migration along the plane of the gel. Blotting electrodes above and below the gel are then energized to impress a potential normal to the gel which causes the separated species to migrate out of the gel onto an adjacent membrane. A plate electrode underneath the gel is simulated by an array of exposed wires, parallel to each other as well as to the separation electrodes.
The cell operates in a separatory mode and a blotting mode, the passage from one to the next being governed by an electric switch arranged such that the field established by the separation electrodes during the separatory mode experiences no interference from the blotting electrodes. When the cell is in the separatory mode, the wires in the array are electrically isolated from each other to permit each to adjust to the voltage of the buffer immediately surrounding it, according to the distance of each wire from the separation electrodes. In the blotting mode, the wires in the array are electrically connected, and their spacing is close enough to simulate, in the region of the gel, an electrical field created by a continuous plate electrode.
Since the upper blotting electrode can be handled without disturbing the gel, it may assume any of a variety of forms. One such form is an array of electrode wires similar to the array beneath the gel. Other possibilities are disclosed below.
A uniform transfer of separated solutes during the blotting phase of the operation is achieved by preventing bubbles generated during blotting from coming to rest against the membrane. Various embodiments of the invention are described for preventing bubbles generated at the lower blotting electrodes from reaching the membrane or from stagnating and accumulating beneath the membrane. A porous plate serves as a support for both the membrane and the gel while permitting full fluid and electrical contact of the membrane and gel with the buffer solution underlying both.
Further features, embodiments and advantages of the invention are described below.